HSV-1 infection in human induced pluripotent stem cell-derived neurons: cellular models for quiescence and drug discovery

Background: Herpes simplex virus, type 1 (HSV-1) establishes latency in human sensory ganglia following primary infection through mucosal tissues. Once latent, the virus persists for the host’s lifetime, with periodic reactivations that cause lytic lesions. First-line medications like acyclovir (ACV) abort lytic reactivations, but drug resistance has been reported and second line drugs may cause serious side effects. These facts, together with inefficacy of antivirals against latency, compel new drug screens; human neural tissues that model aspects of latency are arguably well-suited for such screens. Studies: Study 1. Human induced pluripotent stem cell-derived neurons (iPSC-neurons) were used to model HSV-1 infection employing an HSV-1 construct that incorporates dual fluorescent reporter genes from different kinetic expression groups. Lytic infection was demonstrated initially. ‘Quiescent’ infection was next established using protocols from animal models of latent infection. The quiescent infection fulfilled most criteria for latency, including viral gene expression and heterochromatization patterns. Studies 2 and 3: The antiviral activity of two series of compounds was investigated using monkey epithelial cells and iPSC-neurons. Anti-lytic activity that reduced viral copy number and protein levels was induced by four compounds, of which two compounds also inhibited reactivation of HSV-1 from quiescence/latency. Four compounds were also efficacious against varicella zoster virus and/or human cytomegalovirus infections. Study 4: Moderate throughput platforms for antiviral drug screens based on iPSC-neurons were designed, with readouts based on high content analysis and flow cytometry. Conclusions: Lytic and latent HSV-1 infection was modeled in human iPSC-neurons, with features similar to animal models. Compounds with novel effects against HSV-1 infections were identified using the iPSC-neuron model. Platforms for moderate throughput drug screens are feasible using human iPSC-neuron models. Public Health Significance: Herpesviruses are highly prevalent, with rates exceeding 95% in some populations. Recurrent HSV-1 eye infections are the leading cause of infectious corneal blindness in the USA. HSV-1 encephalitis that is particularly devastating to neonates can be fatal. Associations between HSV-1 seropositivity and cognitive impairment have been reported even without encephalitis. There are no effective HSV-1 vaccines and there are growing concerns about antiviral drug efficacy. In view of the enormous burden, there is a compelling need for novel drug screens

Fine-mapping reveals novel alternative splicing of the dopamine transporter

Center for Human Genetic Research, Massachusetts General Hospital and Department of Neurology, Harvard Medical School, Harvard University, Boston, Massachusetts.Graduate Program in Biology and Biomedical Science, Yale University, New Haven, Connecticut.The dopamine transporter gene (, ) has been implicated in the pathogenesis of numerous psychiatric and neurodevelopmental disorders, including schizophrenia (SZ). We previously detected association between SZ and intronic variants that replicated in two independent Caucasian samples, but had no obvious function. In follow-up analyses, we sequenced the coding and intronic regions of to identify complete linkage disequilibrium patterns of common variations. We genotyped 78 polymorphisms, narrowing the potentially causal region to two correlated clusters of associated SNPs localized predominantly to introns 3 and 4. Our computational analysis of these intronic regions predicted a novel cassette exon within intron 3, designated E3b, which is conserved among primates. We confirmed alternative splicing of E3b in post-mortem human substantia nigra (SN). As E3b introduces multiple in-frame stop codons, the open reading frame is truncated and the spliced product may undergo nonsense mediated decay. Thus, factors that increase E3b splicing could reduce the amount of unspliced product available for translation. Observations consistent with this prediction were made using cellular assays and in post-mortem human SN. In mini-gene constructs, the extent of splicing is also influenced by at least two common haplotypes, so the alternative splicing was evaluated in relation to SZ risk. Meta-analyses across genome-wide association studies did not support the initial associations and further post-mortem studies did not suggest case-control differences in splicing. These studies do not provide a compelling link to schizophrenia. However, the impact of the alternative splicing on other neuropsychiatric disorders should be investigated. © 2010 Wiley-Liss, Inc

How rare and common risk variation jointly affect liability for autism spectrum disorder

Abstract Background Genetic studies have implicated rare and common variations in liability for autism spectrum disorder (ASD). Of the discovered risk variants, those rare in the population invariably have large impact on liability, while common variants have small effects. Yet, collectively, common risk variants account for the majority of population-level variability. How these rare and common risk variants jointly affect liability for individuals requires further study. Methods To explore how common and rare variants jointly affect liability, we assessed two cohorts of ASD families characterized for rare and common genetic variations (Simons Simplex Collection and Population-Based Autism Genetics and Environment Study). We analyzed data from 3011 affected subjects, as well as two cohorts of unaffected individuals characterized for common genetic variation: 3011 subjects matched for ancestry to ASD subjects and 11,950 subjects for estimating allele frequencies. We used genetic scores, which assessed the relative burden of common genetic variation affecting risk of ASD (henceforth “burden”), and determined how this burden was distributed among three subpopulations: ASD subjects who carry a potentially damaging variant implicated in risk of ASD (“PDV carriers”); ASD subjects who do not (“non-carriers”); and unaffected subjects who are assumed to be non-carriers. Results Burden harbored by ASD subjects is stochastically greater than that harbored by control subjects. For PDV carriers, their average burden is intermediate between non-carrier ASD and control subjects. Both carrier and non-carrier ASD subjects have greater burden, on average, than control subjects. The effects of common and rare variants likely combine additively to determine individual-level liability. Limitations Only 305 ASD subjects were known PDV carriers. This relatively small subpopulation limits this study to characterizing general patterns of burden, as opposed to effects of specific PDVs or genes. Also, a small fraction of subjects that are categorized as non-carriers could be PDV carriers. Conclusions Liability arising from common and rare risk variations likely combines additively to determine risk of any individual diagnosed with ASD. On average, ASD subjects carry a substantial burden of common risk variation, even if they also carry a rare PDV affecting risk.http://deepblue.lib.umich.edu/bitstream/2027.42/173934/1/13229_2021_Article_466.pd

Generation of three-dimensional human neuronal cultures: application to modeling CNS viral infections

Abstract Background A variety of neurological disorders including neurodegenerative diseases and infection by neurotropic viruses can cause structural and functional changes in the central nervous system (CNS), resulting in long-term neurological sequelae. An improved understanding of the pathogenesis of these disorders is important for developing efficacious interventions. Human induced pluripotent stem cells (hiPSCs) offer an extraordinary window for modeling pathogen-CNS interactions, and other cellular interactions, in three-dimensional (3D) neuronal cultures that can recapitulate several aspects of in vivo brain tissue. Methods Herein, we describe a prototype of scaffold-free hiPSC-based adherent 3D (A-3D) human neuronal cultures in 96-well plates. To test their suitability for drug screening, A-3D neuronal cultures were infected with herpes simplex virus type 1 (HSV-1) with or without acyclovir. Results The half maximal inhibitory concentration (IC50) of acyclovir was 3.14 μM and 3.12 μM determined using flow cytometry and the CX7 High Content Screening platform, respectively. Conclusions Our A-3D neuronal cultures provide an unprecedented opportunity for high-content drug screening programs to treat human CNS infections

iPSC Neuronal Assay Identifies Amaryllidaceae Pharmacophore with Multiple Effects against Herpesvirus Infections

The Amaryllidaceae alkaloid <i>trans</i>-dihydrolycoricidine <b>7</b> and three analogues <b>8</b>–<b>10</b> were produced via asymmetric chemical synthesis. Alkaloid <b>7</b> proved superior to acyclovir, the current standard for herpes simplex virus, type 1 (HSV-1) infection. Compound <b>7</b> potently inhibited lytic HSV-1 infection, significantly reduced HSV-1 reactivation, and more potently inhibited varicella zoster virus (VZV) lytic infection. A configurationally defined (3<i>R</i>)-secondary alcohol at C3 proved crucial for efficacious inhibition of lytic HSV-1 infection